New ellipticine compounds

ABSTRACT

The invention relates to a compound selected from those of formula (I): ##STR1## wherein R 1 , R 2 , R 3 , R 4  and R 5  are as defined in the specification, and medicaments.

The present invention relates to new ellipticine compounds, a processfor their preparation and pharmaceutical compositions containing them.The compounds of the present invention are of valuable therapeutic useowing to their anti-tumour activity.

Compounds of the ellipticine family are already known for theiranti-cancer properties. For example, the patent application EP-A-42348may be mentioned in which olivacine compounds were studied. The patentapplications EP-A-393575 and EP-A-10029 describe pyrido[4,3-b]carbazoleor ellipticine compounds.

Therapeutic needs require the constant development of new anti-canceragents with the aim of obtaining molecules that are both more active andbetter tolerated.

The present invention relates to ellipticine compounds which exhibitnovelty compared with the compounds described in the prior art: theintensity of the pharmacological properties, that is to say theanti-tumour activity of the compounds of the invention, has beenoptimised by inserting an aminoalkylaminocarbonyl group into the1-position of 6H-pyrido[4,3-b]carbazole.

In addition, the compounds of the present invention are distinguished bytheir greater cytotoxicity in vitro as well as their improved activityin vivo compared with the reference compounds.

The present invention relates more especially to compounds of thegeneral formula (I): ##STR2## wherein:

R₁ represents the radical ##STR3## in which: n is an integer of 1 to 6inclusive,

R, R' and R₆, which are the same or different, are selected,independently of one another, from hydrogen and straight-chain orbranched alkyl containing 1 to 6 carbon atoms inclusive optionallysubstituted by one or more hydroxy, or R and R', together with thenitrogen atom to which they are attached, form a heterocycle optionallycontaining a second hetero atom, which heterocycle may be substituted byone or more straight-chain or branched alkyl containing 1 to 6 carbonatoms inclusive, and R₆ is as defined above, or R and R₆ together form aheterocycle which is unsubstituted or substituted by one or morestraight-chain or branched alkyl containing 1 to 6 carbon atomsinclusive, and R' is as defined above,

R₂ represents straight-chain or branched alkyl containing 1 to 6 carbonatoms inclusive,

R₃, R₄ and R₅, which are the same or different, are selected,independently of one another, from hydrogen and straight-chain orbranched alkyl containing 1 to 6 carbon atoms inclusive,

their optical isomers, their possible N-oxides andpharmaceutically-acceptable addition salts with an acid or base.

The heterocycles formed by the radicals R and R' together with thenitrogen atom to which they are attached include more especiallypyrrole, pyrrolines, pyrrolidine, imidazole, imidazolines,imidazolidine, pyrazole, pyrazolines, oxazole, oxazolines, oxazolidine,pyridine, piperidine, pyridazine, pyrimidine, pyrazine, piperazine,morpholine and thiomorpholine.

The invention extends also to a process for the preparation of compoundsof formula (I) which is characterised by a first biomimetic reactionaccording to Besselievre Husson (Tetrahedron, (1981), 37, 241-246) inwhich a compound of formula (II) is reacted in an acid medium, forexample in acetic acid, with a compound of formula (III): ##STR4##wherein R₂ and R₅ are as defined for formula (I) and R'₄ represents astraight-chain or branched alkyl radical containing from 1 to 6 carbonatoms, to obtain, after debenzylation followed by heating in thepresence of a dialkyl oxalate, a compound of formula (IV): ##STR5##wherein R₂, R'₄ and R₅ are as defined hereinbefore and R₀ represents analkyl group containing from 1 to 5 carbon atoms, which may be cyclisedby heating at reflux in the presence of POCl₃ in toluene so as to yield,after dehydrogenation on palladium-on-carbon, a compound of formula(Va): wherein R₀, R₂, R'₄ and R₅ are as defined hereinbefore, of which,if desired, the 6-nitrogen atom of the carbazole is substituted bytreatment with a dialkyl carbonate of formula ##STR6## wherein R'₃represents a straight-chain or branched alkyl radical containing from 1to 6 carbon atoms, in a polar solvent, such as dimethylformamide, in thepresence of an alkali metal carbonate, such as potassium carbonate, anda crown ether, to obtain a compound of formula (Vb): ##STR7## whereinR₂, R'₃, R'₄, R₅ and R₀ are as defined hereinbefore, the compounds offormulae (Va) and (Vb) forming the totality of the compounds of formula(V): ##STR8## wherein R₂, R₃, R'₄, R₅ and R₀ are as definedhereinbefore, which compounds of formula (V) are subjected to asubstitution reaction with a compound of formula (VI): ##STR9## whereinR, R', R₆ and n are as defined for formula (I), to obtain compounds offormula (Ia): ##STR10## wherein R₁, R₂, R₃, R'₄ and R₅ are as definedhereinbefore, which compounds of formula (Ia) may be dealkylated by theaddition of a boron trihalide, such as boron tribromide, to yieldcompounds of formula (Ib): ##STR11## wherein R₁, R₂, R₃ and R₅ are asdefined hereinbefore, the compounds of formula ( Ia ) and ( Ib ) formingthe totality of the compounds of the general formula (I) which, whereappropriate, may be purified according to a conventional separationtechnique and, if desired, converted into their possible N-oxides andpharmaceutically acceptable addition salts with an acid or base.

The dealkylation reaction described for the conversion of a compound offormula (Ia) into a compound of formula (Ib) may also be carried outdirectly on the ester of formula (V) described hereinbefore, so as toobtain a compound of formula (V'): ##STR12## wherein R₂, R₃, R₅ and R₀are as defined hereinbefore, which is then treated with a compound offormula (VI) as defined hereinbefore, under the same operationalconditions as defined for the compound of formula (V), to yield acompound of formula (Ib) as defined hereinbefore.

The compounds of formula (I) possess valuable pharmacologicalproperties. Apart from their greater cytotoxicity in vitro, they proveto be more effective and at least as active in vivo as the compoundstaken as reference compounds.

In addition, the compounds described in the present invention are activeat very low doses and result in numerous survivors at the optimum doses.They therefore have an excellent therapeutic index.

Owing to their ability to be inserted into DNA and to cause cleavages byinhibiting the topoisomerases, they can be used therapeutically asanti-tumour agents.

The present invention relates also to pharmaceutical compositionscontaining the compounds of formula (I), their N-oxides, their opticalisomers or one of their pharmaceutically-acceptable addition salts witha base or acid, alone or in combination with one or more inert non-toxicexcipients or carriers.

Of the pharmaceutical compositions according to the invention there maybe mentioned more especially those which are suitable for oral,parenteral, nasal, rectal, perlingual, ocular or respiratoryadministration, and especially tablets or dragees, sublingual tablets,sachets, paquets, gelatin capsules, glossettes, lozenges, suppositories,creams, ointments, dermal gels, injectable or drinkable preparations,aerosols and eye or nose drops.

The dosage used varies in accordance with the age and weight of thepatient, the administration route, and the nature of the therapeuticindication and possible associated treatments, and ranges from 0.1 to400 mg per day in one or more administrations.

The following Examples illustrate the invention but do not limit it inany way. The starting materials are known or are prepared by knownmethods of operation.

Preparation of 2-(β-benzylamino)ethyl-6-methoxy-1-methylcarbazole

5-methoxyindole and1-benzyl-4-(1,1-ethylenedioxyethyl)-1,2,3,6-tetrahydropyridine (boilingpoint:184°-188° C. at 10 mm Hg, obtained by the reduction of1-benzyl-4-(1,1-ethylenedioxyethyl)-pyridinium chloride with sodiumborohydride in methanol) are heated at reflux for 66 hours in 50% aceticacid. The mixture is then poured into 2 liters of water. Afterextraction with dichloromethane and removal of the solvent byevaporation, an oily residue is obtained which is subsequently taken upin ethyl acetate. The solid progressively formed is filtered off andwashed with the necessary minimum of cold dichloromethane. Colourlessacetate crystals are obtained. The corresponding base crystallises intoluene to yield colourless crystals.

Melting point: 150° C.

EXAMPLE 11-(N,N-Dimethylaminoethylaminocarbonyl)-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazoleStep A 1-Methyl-2-[(β-ethoxalamido)ethyl]-6-methoxycarbazole

35.6 g (88 mmol) of 2-[(β-benzylamino)ethyl]-6-methoxy-1-methylcarbazole(in the form of the acetate) are dissolved in 400 ml of acetic acid towhich 10 g of 10% palladium-on-carbon are added. The mixture is heatedto 50° C. and stirred under hydrogen at normal pressure whilemaintaining the temperature until the theoretical volume of hydrogen hasbeen consumed (10 hours). The solvent is evaporated off under reducedpressure; the residue obtained is taken up in 200 ml of water. Afterfiltration and addition of ammonium hydroxide, the solid is dried in airand recrystallised from toluene. 18.16 g of beige-coloured crystals areobtained.

Yield: 81.4% Melting point: 167°-168° C.

The solid is heated at 100°-110° C. for 1 hour in 70 ml of diethyloxalate and the mixture obtained is evaporated to dryness. The solidresidue is taken up in cyclohexane, filtered and recrystallised fromethyl acetate to yield 23.92 g of the desired compound.

Yield: 94.6% Melting point: 144° C.

    ______________________________________                                        Elemental analysis: C.sub.20 H.sub.22 N.sub.2 O.sub.4                                   C %        H %    N %                                               ______________________________________                                        calculated  67.78        6.26   7.91                                          found       67.96        6.35   8.21                                          ______________________________________                                    

Step B Ethyl 1- (3,4-dihydro-9-methoxy-5-methyl-6H-pyrido [4,3-b]carbazolyl)carboxylate

3.54 g (10 mmol) of the amide obtained in Step A are dissolved in 300 mlof boiling toluene and treated dropwise with 30 ml of POCl₃. Reflux ismaintained for 24 hours. The solvent is evaporated under reducedpressure and the residue is then taken up in 200 ml of water. Thesolution is filtered and adjusted to pH 9-10 by the addition of sodiumcarbonate. The resulting solid is washed with water, dried andrecrystallised from ethyl acetate. 2.36 g of yellow crystals areobtained.

Yield: 70%. Melting point: 233°-234° C.

    ______________________________________                                        Elemental analysis: C.sub.20 H.sub.20 N.sub.2 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  71.41        5.99   8.33                                          found       71.39        5.81   8.11                                          ______________________________________                                    

Step C Ethyl1-(9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazolyl)carboxylate

2.036 g (6 mmol) of the ester obtained in the preceding Step are heatedat reflux for 10 minutes in 40 ml of diphenyl ether in the presence of300 mg of 10% palladium-on-carbon, or for 45 minutes in decaline in thepresence of 200 mg of 10% Pd-on-carbon, or for 5 hours in mesitylene inthe presence of the same amounts of dehydrogenation agent. The residueis extracted with 1N hydrochloric acid, and the aqueous solution is thenneutralised with sodium bicarbonate. The precipitate obtained isextracted with dichloromethane. After evaporation of the solvent, thesolid residue remaining is purified by chromatography on a column ofsilica gel. Elution with dichloromethane results in traces of diphenylether. The desired compound is eluted with a 98/2 mixture ofdichloromethane and ethyl acetate. The product is recrystallised fromethyl acetate, 1.17 g of yellow crystals are obtained.

Yield: 58.3% Melting point: 255° C.

    ______________________________________                                        Elemental analysis: C.sub.20 H.sub.18 N.sub.2 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  71.84        5.43   8.38                                          found       71.75        5.54   8.45                                          ______________________________________                                    

Step D Ethyl1-(5,6-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazolyl)carboxylate

A mixture of 334 mg (1 mmol) of the ester obtained in the precedingStep, 250 mg of dry finely pulverised potassium carbonate, 5 ml ofdimethyl carbonate, 1 ml of dimethylformamide and 1 drop of crown ether18C6 is heated at reflux, with stirring, for 8 hours. After evaporationto dryness the residue is taken up in water. The solid obtained is driedin air and then recrystallised from cyclohexane. 270 mg of yellowcrystals are obtained.

Yield: 77.5% Melting point: 162°-164° C.

    ______________________________________                                        Elemental analysis: C.sub.21 H.sub.20 N.sub.2 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  72.39        5.79   8.04                                          found       72.16        5.91   8.03                                          ______________________________________                                    

Step E1-(N,N-Dimethylaminoethylaminocarbonyl)-5,6-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazole

348 mg (1 mmol) of the ester obtained in the preceding Step are heatedin 5 ml of N,N-dimethyl-1,2-diaminoethane for 18 hours. Excess amine isevaporated off under reduced pressure. The residue obtained is taken upin water and the resulting solid is dried in air and recrystallised fromcyclohexane. 234 mg of yellow crystals are obtained.

Yield: 60% Melting point: 139° C.

    ______________________________________                                        Elemental analysis: C.sub.23 H.sub.26 N.sub.4 O.sub.2                                   C %        H %    N %                                               ______________________________________                                        calculated  70.74        6.71   14.35                                         found       70.53        6.96   14.35                                         ______________________________________                                    

Step F1-(N,N-Dimethylaminoethylaminocarbonyl)-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

390 mg (1 mmol) of the compound obtained in Step E are dissolved in 40ml of dry dichloromethane under an argon atmosphere. The solution iscooled to -70° C. 10 molar equivalents of boron tribromide, that is 10ml of a commercial 1M solution in dichloromethane, are added dropwise.The mixture is allowed to return to room temperature (18 hours), and isthen poured into 100 ml of iced water. The solution is adjusted to abasic pH by the addition of triethylamine, and then stirred for 3 hoursat room temperature. The product is extracted with dichloromethane andthe solvent is evaporated off for the purpose of carrying outchromatography on a column of alumina (eluant dichloromethane/ethanol95/5). The final product crystallises from ethyl acetate which isevaporated off. 164 mg of yellow crystals are obtained.

Yield: 43.6% Melting point: 256° C.

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.24 N.sub.4 O.sub.2                                   C %        H %    N %                                               ______________________________________                                        calculated  70.18        6.43   14.88                                         found       69.79        6.69   14.49                                         ______________________________________                                    

Example 21-(N,N-Dimethylaminopropylaminocarbonyl)-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 for Steps A to D.

Step E1-(N,N-Dimethylaminopropylaminocarbonyl)-5,6-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazole

In Step E the heating time for the ester is set at 8 hours and thediamine used is N,N-dimethyl-1,3-diaminopropane. The product obtained isextracted with dichloromethane; the solvent is then evaporated off andthe residue is treated with excess maleic acid in acetone to obtain thecorresponding salt.

Step F

1-(N,N-Dimethylamincpropylaminocarbonyl)-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

404 mg (1 mmol) of the amide obtained in the preceding Step aredissolved in 40 ml of dry dichioromethane under an argon atmosphere. Thesolution is cooled to -70° C. 10 molar equivalents of boron tribromideare added dropwise; the mixture is left at room temperature for 18 hoursand then poured into 100 ml of iced water. The solution is renderedbasic by the addition of triethylamine and stirred at room temperaturefor 3 hours. The product is extracted with dichloromethane; evaporationof the solvent yields a residue which crystallises in ethyl acetate.

Yield: 76.8% Melting point: 198° C.

    ______________________________________                                        Elemental analysis: C.sub.23 H.sub.26 N.sub.4 O.sub.2, 0.5 H.sub.2 O                    C %        H %    N %                                               ______________________________________                                        calculated  69.15        6.81   14.02                                         found       69.55        6.80   13.94                                         ______________________________________                                    

Example 3

1-(N,N-Dimethylaminoethylaminocarbonyl)-5,6-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of Step F.

Yield: 60% Melting point: 139° C.

    ______________________________________                                        Elemental analysis: C.sub.23 H.sub.26 N.sub.4 O.sub.2                                   C %        H %    N %                                               ______________________________________                                        calculated  70.74        6.71   14.35                                         found       70.53        6.96   14.35                                         ______________________________________                                    

Example 41-(N,N-Dimethylaminopropylaminocarbonyl)-5,6-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 2 with the exception of Step F.

Yield: 92.5% Melting point: 139° C.

    ______________________________________                                        Elemental analysis: C.sub.24 H.sub.28 N.sub.4 O.sub.2, 2C.sub.4 H.sub.4       O.sub.4                                                                                 C %        H %    N %                                               ______________________________________                                        calculated  60.37        5.70   8.80                                          found       60.28        5.62   9.03                                          ______________________________________                                    

Example 5 1-(N,N-Dimethylaminopropylaminocarbonyl)-5-methyl-9-methoxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of Steps D and F, which arenot carried out, and with the following modifications to Step E: theamine used is N,N-dimethyl-1,3-diaminopropane, and the solid, afterhaving been dried, is recrystallised from ethyl acetate.

Yield: 93% Melting point: 206° C.

    ______________________________________                                        Elemental analysis: C.sub.23 H.sub.26 N.sub.4 O.sub.2, H.sub.2 O                        C %        H %    N %                                               ______________________________________                                        calculated  67.72        6.91   13.72                                         found       67.60        6.72   14.05                                         ______________________________________                                    

Example 61-(N,N-Dimethylaminoethylaminocarbonyl)-5-methyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of the intermediate Step D andwith the following modifications to Steps E and F:

Step E

the heating time is set at 8 hours,

Step F

as the compound obtained after stirring for 3 hours at room temperatureis insoluble in dichloromethane, it is filtered off and recrystallisedfrom ethyl acetate.

Yield: 47% Melting point: >270° C.

    ______________________________________                                        Elemental analysis: C.sub.21 H.sub.22 N.sub.4 O.sub.2                                   C %        H %    N %                                               ______________________________________                                        calculated  69.59        6.12   15.46                                         found       69.40        6.14   15.21                                         ______________________________________                                    

Example 71-(N,N-Dimethylaminopropylaminocarbonyl)-5-methyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of the intermediate Step D andwith the following modifications:

Step E

the amine used is N,N-dimethyl-1,3-diaminopropane, and the solidobtained is dried in air and recrystallised from ethyl acetate,

Step F

the product obtained after stirring for 3 hours is filtered off andrecrystallised from ethyl acetate.

Yield: 56% Melting point: 218° C.

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.24 N.sub.4 O.sub.2, 0.5 H.sub.2 O                    C %        H %    N %                                               ______________________________________                                        calculated  68.57        6.49   14.55                                         found       68.45        6.43   14.86                                         ______________________________________                                    

Example 81-(N,N-Dimethylaminoethylaminocarbonyl)-5-methyl-9-methoxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of Steps D and F and with thefollowing modification to Step E: The heating time is set at 8 hours.

Yield: 88% Melting point: 215° C.

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.24 N.sub.4 O.sub.2, 0.5 H.sub.2 O                    C %        H %    N %                                               ______________________________________                                        calculated  68.57        6.49   14.54                                         found       68.64        6.53   14.48                                         ______________________________________                                    

Example 91-[2-(Pyrrolidin-1-yl)ethylaminocarbonyl]-5-methyl-9-methoxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 1 with the exception of Steps D and F, which arenot carried out, and the amine used in Step E, which is2-(pyrrolidin-1-yl)-1-aminoethane.

Example 101-[2-(Pyrrolidin-1-yl)ethylaminocarbonyl)-5-methyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

This compound is prepared in accordance with the same protocol as thatdescribed in Example 6 using 2-(pyrrolidin-1-yl)-1-aminoethane in StepE.

Example 11 1-{2-[(2-Hydroxyethyl)amino]ethylaminocarbonyl}-5,6-dimethyl-9-hydroxy-6H-pyrido -[4,3-b]carbazole Step A Ethyl1-(5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazolyl)carboxylate

The ester obtained in Step D of Example 1 is used in the demethylationreaction described in Step F of Example 1 to obtain1-ethoxycarbonyl-5,6-dimethyl-9-hydroxy-6H-pyrido [4,3-b]carbazole.

Step B1-{2-[(2-Hydroxyethyl)amino]ethylaminocarbonyl}-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

0.4 g (1.198 mmol) of the ester obtained in the preceding Step aredissolved in 10 ml of 2-(2-aminoethylamino)ethanol under an argonatmosphere. The mixture is heated at 120° C. for 24 hours.

Excess amine which has not reacted is removed by placing under a vacuum.Water and dichloromethane are then added to the reaction mixture. Thecustomary treatment of the organic phases yields an oily residue whichis taken up in ethanol, and then poured into a saturated solution ofethanol in gaseous hydrogen chloride. The evaporation of the ethanolcauses the crystallisation of 0.58 g of the dihydrated dihydrochlorideof the desired compound in the form of red crystals.

Yield: 97%

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.24 O.sub.3 N.sub.4, 2HCl, 2H.sub.2 O                 C %  H %        N %    Cl %                                         ______________________________________                                        calculated  52.70  6.03       11.17                                                                              14.14                                      found       52.55  5.79       10.98                                                                              17.37                                      ______________________________________                                    

Example 121-[(4-Aminobutyl)aminocarbonyl]-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

0.47 g (1.407 mmol) of the ester obtained in Step A of Example 11 aredissolved in 9 ml of 1,4-diaminobutane under an argon atmosphere. Themixture is heated at 130° for 3 hours.

Excess amine which has not reacted is removed by placing under a vacuum.Water and dichloromethane are then added to the reaction mixture.

The major portion of the insoluble product is isolated bysuction-filtering and dried. The other portion is obtained by extractionwith dichloromethane and the extract is dried over magnesium sulfate,filtered and then evaporated to dryness.

The total product is recrystallised from absolute ethanol to yield 0.34g of orange crystals corresponding to the desired compound.

Yield: 62% Melting point: >95° C. (decomposition)

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.24 N.sub.4 O.sub.2, 0.8 H.sub.2 O                    C %        H %    N %                                               ______________________________________                                        calculated  67.60        6.60   14.33                                         found       67.85        6.98   13.02                                         ______________________________________                                    

Example 131-[N-(2-Pyrrolidin-1-ylethyl)aminocarbonyl]-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

0.4 g (1.198 mmol) of the ester obtained in Step A of Example 11 aredissolved in 7.5 ml of 1-(2-aminoethyl)pyrrolidine under an argonatmosphere. The mixture is heated at 120° C. for 16 hours.

Excess amine is removed by placing under a vacuum, and the reactionmixture is hydrolysed. The customary treatment of the organic phaseyields 0.341 g of yellow crystals corresponding to the desired compound.

Yield: 65% Melting point: >120° C. (decomposition)

    ______________________________________                                        Elemental analysis: C.sub.24 H.sub.26 N.sub.4 O.sub.2, 2H.sub.2 O                       C %        H %    N %                                               ______________________________________                                        calculated  66.03        6.97   12.84                                         found       65.99        7.11   12.27                                         ______________________________________                                    

Example 141-[N-(3-Morpholinopropyl)aminocarbonyl]-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

0.36 g (1. 078 mmol) of the ester obtained in Step A of Example 11 aredissolved in 10 ml of 4-(3-aminopropyl) morpholine under an argonatmosphere. The mixture is heated at 120° C. for 19 hours.

Excess amine which has not reacted is removed by placing under a vacuum.The reaction mixture is hydrolysed and taken up in dichloromethane.

The mixture is stirred for approximately 1 hour.

The major portion of the insoluble product is isolated bysuction-filtering and dried.

The other portion is extracted with dichloromethane and the extract isdried over magnesium sulfate, filtered and evaporated to dryness.

The total product is recrystallised from absolute ethanol to yield 0.42g of yellow crystals corresponding to the desired compound.

Yield: 90% Melting point: 110° C.

    ______________________________________                                        Elemental analysis: C.sub.25 H.sub.28 N.sub.4 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  69.42        6.53   12.95                                         found       69.34        6.50   12.86                                         ______________________________________                                    

Example 151-[(N,N-Dimethyl-2-aminoethyl)aminocarbonyl]-5-methyl-6-ethyl-9-hydroxy-6H-pyrido4,3-b]carbazoleStep A 1 -Ethoxycarbonyl-5-methyl-6-ethyl-9-methoxy-6H-pyrido[4,3-b]carbazole

To a mixture of 5 g (15 mmol) of the ester obtained in Step C of Example1, in 10 ml of dimethylformamide, are added 142 ml of diethyl carbonate,3.75 g of potassium carbonate and 200 mg of crown ether 18C6 under anargon atmosphere. The mixture is heated at 130°-140° C. during 6 daysand concentrated. Water and dichloromethane are then added to thereaction mixture. The customary treatment of the organic phase yields4.11 g of yellow crystals from toluene.

Yield: 76% Melting point: 149° C.

    ______________________________________                                        Elemental analysis: C.sub.22 H.sub.22 N.sub.2 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  72.91        6.12   7.73                                          found       73.06        6.19   7.72                                          ______________________________________                                    

Step B1-Ethoxycarbonyl-9-hydroxy-5-methyl-6-ethyl-6H-pyrido[4,3-b]carbazole

4 g (11.05 mmol) of the ester obtained in the preceding Step aredissolved in 440 ml of dried dichloromethane, under an argon atmosphere,at -78° C. 120 ml (110.5 mmol) of boron tribromide (1M solution indichloromethane) are added. The mixture is stirred for 2 hours and 30minutes at -78° C. and then at room temperature during one hour. Excessboron tribromide is hydrolysed by adding drops of water maintaining thetemperature at c:a 0° C. The solution is adjusted to a basic pH by theaddition of aqueous ammonia and stirred for another 1 hour. The solidobtained is isolated, dried and the organic phase treated in a customaryway. The crude product is recrystallised from toluene to yield 2.98 g ofthe desired compound.

Yield: 78% Melting point: 260° C.

    ______________________________________                                        Elemental analysis: C.sub.21 H.sub.20 N.sub.2 O.sub.3                                   C %        H %    N %                                               ______________________________________                                        calculated  72.40        5.79   8.04                                          found       72.94        5.99   7.93                                          ______________________________________                                    

Step C1-[(N,N-Dimethyl-2-aminoethyl)aminocarbonyl]-5-methyl-6-ethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole

The mixture formed by 0.8 g (2.30 mmol) of the ester obtained in thepreceding Step in 12 ml of N,N-dimethylaminoethylenediamine under anargon atmosphere is heated at 115° C. for 36 hours. The mixture is thenhydrolysed, subsequently extracted with dichloromethane, and the organicphase is dried over magnesium sulfate and then filtered. Afterevaporation of the dichloromethane, the oily residue is purified bychromatography on a column of silica gel (eluant dichloromethane/ethanol9:1 and approximately 0.5% triethylamine).

0.424 g of yellow crystals corresponding to the desired compound isobtained.

Yield: 44% Melting point: >115° C. (decomposition)

    ______________________________________                                        Elemental analysis: C.sub.23 H.sub.26 N.sub.4 O.sub.2, 1.5 H.sub.2 O                    C %        H %    N %                                               ______________________________________                                        calculated  66.17        7.00   13.42                                         found       66.01        6.81   13.41                                         ______________________________________                                    

Pharmacological Study

The following Examples make it possible to demonstrate that thetherapeutic index of the better compounds of the invention is excellent:

they are cytotoxic,

they are active from doses of less than 5 mg/kg up to 30 to 60 mg/kg,and

they produce numerous survivors at the optimum doses.

Example A Cytotoxicity of the compounds and of the reference compounds

Seven cell lines were used:

2 murine leukaemias, L1210 and P388;

1 human epidermoid carcinoma, KB-3-1, and a human pulmonary carcinomaS1;

the corresponding resistant lines: KB-A1, the multidrug resistance ofwhich was induced by adriamycin (ADR) on KB-3-1; P388/VCR-20, themultidrug resistance of which was induced by vincristine (VCR) on P388 ;and S1/tMDR, the multidrug resistance of which was obtained bytransfecting the human MDR1 gene into S1 cells.

The cells are cultured in complete culture medium RPMI 1640 containing10% foetal calf serum, 2 mM glutamine, 50 units/ml of penicillin, 50μg/ml of streptomycin and 10 mM HEPES.

The cells are distributed on microplates and exposed to the cytotoxiccompounds. The cells are then incubated for approximately 4 periods ofcell doubling, that is 2 days (P 388, P388/VCR-20 and L1210) and 4 days(KB-A1, KB-3-1, S1 and S1/tMDR). The number of viable cells is thenquantified by a colorimetric assay, the Microculture Tetrazolium Assay(Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D. and Mitchell J.R., Evaluation of a tetrazolium-based semiautomated colorimetric assay :assessment of chemosensitivity testing, Cancer Res., 47, 936-942, 1987).The results are expressed as IC₅₀, the cytotoxic concentration whichinhibits the proliferation of tumour cells by 50%. The results obtainedfor line L1210 are shown in Table 1:

                  TABLE 1                                                         ______________________________________                                        In vitro activity- Line L1210                                                                 Cytotoxicity                                                  Compounds       IC.sub.50, nM                                                 ______________________________________                                        ex 1             2,6                                                          ex 2             4,3                                                          ex 3            574,3                                                         ex 4            874,7                                                         ex 5            1495,0                                                        ex 6             3,1                                                          ex 7            41,0                                                          ex 8            487,0                                                         ex 9            1495,5                                                        ex 11           29,7                                                          ex 12           449,8                                                         ex 13           13,1                                                          ex 14           10,4                                                          ADR             26,4                                                          elliptinium acetate                                                                           138,9                                                         ______________________________________                                    

The results show that the compounds of Examples 1, 2 and 6 areapproximately 9 times more cytotoxic than adriamycin and 46 times morecytotoxic than elliptinium acetate.

Example B Cytotoxicity of the compounds and the reference compoundsagainst lines exhibiting the multidrug resistance phenotype (MDR)

The resistance factor is defined by the ratio: ##EQU1##

                  TABLE 2                                                         ______________________________________                                        Cytotoxicities and resistance factors                                                   Cytotoxicity, IC.sub.50 (nM) resistance factor                                                             Elliptinium                            Lines       ex 1   ex 2    ex 6  ADR   acetate                                ______________________________________                                        sensitive P388                                                                             5     11      19    20    --                                     resistant P388/                                                                            4     38      10    340   --                                     VCR-20                                                                        resistance factor                                                                         0,8    3,4     0,5   17    --                                     sensitive KB-3-1                                                                          12     19      20     5     226                                   resistant KB-A1                                                                           120    1217    737   6869  4322                                   resistance factor                                                                         10     64      37    1374   19                                    sensitive S1                                                                              49     57      42    36    --                                     resistant S1/tMDR                                                                         25     38      29    135   --                                     resistance factor                                                                         0,5    0,7     0,7   3,8   --                                     ______________________________________                                    

The increased resistance of the resistant lines to the compounds is farweaker than the resistance to adriamycin. The activity of the compoundsagainst KB-A1 is far greater than that of adriamycin or elliptiniumacetate. The resistant lines P388/VCR-20 and S1/tMDR (the resistance toADR of which is weaker) are more sensitive to Examples 1 and 6 than thecorresponding sensitive lines. Those compounds could therefore be usedagainst tumours that are resistant to adriamycin.

Example C In vivo activity Example C-1 Anti-tumour activity of thecompounds against leukaemia P388

Line P388 (murine leukaemia) was supplied by the National CancerInstitute (NCI) (Frederick, USA). The tumour cells (10⁶ cells) wereinoculated on day 0 into the peritoneal cavity of female BDF1 mice(Iffa-Credo, France). Eight to ten mice, of a weight ranging from 18 to20 g, are used for each experimental group.

The compounds were administered on day 1 via the intraperitoneal routeat the doses indicated.

The anti-tumour activity is expressed as a T/C %: ##EQU2##

Animals that have survived 60 days (long-term survivors) are indicated.

The results are shown in the following Table 3:

                  TABLE 3                                                         ______________________________________                                        In vivo activity: leukaemia P388                                                                              Survivors on                                               Dose      T/C      d60/total no.                                 Compounds    (mg/kg)   (%)      of mice                                       ______________________________________                                        Example 1     5        163      1/10                                                       10        183      1/10                                                       20        220      2/10                                                       30        200      3/10                                                       60        257      3/10                                          Example 2     5        177      0/10                                                       10        190      0/10                                                       20        250      3/10                                                       30        204      1/10                                                       40        219      2/10                                                       50        222      3/10                                                       60        248      3/10                                          Example 6     5        280      4/10                                                       10        233      3/10                                                       20        >600     5/10                                                       30        >600     7/10                                          ADR          10        290      2/10                                          elliptinium acetate                                                                         5        171      0/10                                          ______________________________________                                    

The compounds were tested at equitoxic doses. The gravimetric variation,at the optimum dose used for the compounds, is from -0.4 to -0.7 g,whilst it is -0.7 g for adriamycin and -4 g for elliptinium acetate.

The compounds are active at 5 mg/kg and above, all the doses employedgiving long-term survivors for two compounds (Examples 1 and 6 of Table2). The compound of Example 1 used in a single administration of 30mg/kg cures 7 out of 10 treated mice whilst adriamycin cures only 2 andelliptinium acetate cures none. The three compounds tested areconsiderably more active than elliptinium acetate which, moreover, istoxic at 5 mg/kg and above.

Example C-2 Anti-tumour activity of the compounds against Lewispulmonary carcinoma

Lewis pulmonary carcinoma (supplied by NCI, Frederick, USA) was insertedin the form of fragments on d0 and by the subcutaneous route into femaleB6D2F1 mice weighing 18 to 20 g. The compounds were administered at thedoses indicated by the i.v. route on d3, d6 and d9.

The anti-tumour activity was determined on d20 by measuring the tumourvolume: ##EQU3## and by the prolongation of the survival of the treatedgroups, expressed as T/C %. The long-term survivors (d90 ) are recorded.##EQU4##

The compound of Example 1 is very active against this very resistantmodel. A dose of 40 mg/kg is curative for 100% of the animals whilstadriamycin, at the maximum tolerated dose, is far less active (Table 4).

                  TABLE 4                                                         ______________________________________                                        Anti-tumour activity of the compounds against Lewis                           carcinoma                                                                                            median   median  Survi-                                Com-   Dose            T/C, %   T/C, %  vors on                               pound  (mg/kg)  route  (volume) d20                                                                           (survival)                                                                            d90                                   ______________________________________                                        Control --      i.v.   100      100     1/25                                  ex. 1   20      i.v.   1        >300    6/10                                          40      i.v.   0        >300    10/10                                         60      i.v.   0        >300    6/10                                  ADR     2,5     i.v.   63       108     0/10                                           5      i.v.   44       125     2/10                                          10      i.v.   1        128     3/10                                  ______________________________________                                    

Example C-3 Anti-tumour :activity of the compounds against M5076 sarcoma

M5076 sarcoma is supplied by NCI (Frederick, USA). The tumour cells (10⁷cells/animal) were inoculated on d0 into the peritoneal cavity of femaleB6D2F1 mice. The compounds were administered by the i.p. route on d1,d5, d9 and d13 at the doses indicated. The surviving animals on d90 wererecorded and the T/C survival (%) was calculated.

The compound of Example 1, at a dose of 20 mg/kg, is very active againstthat tumour. 6 survivors out of 10 are observed on d90. Elliptiniumacetate is not very active at the maximum tolerated dose (2 mg/kg)--noanimal has survived. The results are reproduced in Table 5.

                  TABLE 5                                                         ______________________________________                                        Anti-tumour activity of the compounds against M5076                           sarcoma.                                                                               Dose             median T/C, %                                                                           Survivors                                 Compound (mg/kg)  route   (survival)                                                                              on d90                                    ______________________________________                                        Control  --       i.p.    100       0/32                                      ex. 1    5        i.p.    168       0/10                                               10       i.p.    301       4/10                                               20       i.p.    >352      6/10                                      ADR      5        i.p.    320       4/10                                      ellipt. ac.                                                                            1        i.p.    153       1/10                                               2        i.p.    180       0/10                                      ______________________________________                                    

Example C-4 Anti-tumour activity of the compounds against colon 38

Colon 38 (supplied by NCI, Frederick, USA) was inserted in the form offragments by the subcutaneous route into female B6D2F1 mice. Thecompounds were administered by the i.v. route on d2 and d9 and theanti-tumour activity was determined on d25 by measuring the tumourvolume (T/C volume, %).

The compound of Example 2, administered by the i.v. route is very activeagainst that solid tumour at a dose of 20 mg/kg and above (T/C=0%)(Table 6). It is as active as 5-fluorouracyle (5-Fu) used at a dose of80 mg/kg.

                  TABLE 6                                                         ______________________________________                                        Anti-tumour activity of the compounds against colon 38                                  Dose                median T/C, %                                   Compound  (mg/kg)     Route   (tumour volume)                                 ______________________________________                                        ex. 1     20          i.v.    41                                                        40          i.v.    16                                                        60          i.v.    0                                               ex. 2     20          i.v.    0                                                         40          i.v.    0                                                         60          i.v.    0                                               ex. 6     10          i.v.    35                                                        20          i.v.    22                                                        30          i.v.    0                                               ADR       15          i.v.    0                                               5-Fu      80          i.v.    0                                               ______________________________________                                    

We claim:
 1. A compound selected from those of formula (I): ##STR13##wherein: R₁ represents the group ##STR14## in which: n is an integer of1 to 6 inclusive,R, R' and R₆, which are the same or different, areselected, independently of one another, from hydrogen and straight-chainor branched alkyl having 1 to 6 carbon atoms inclusive optionallysubstituted by one or more hydroxy, R₂ represents straight-chain orbranched alkyl having 1 to 6 carbon atoms inclusive, R₃, R₄ and R₅,which are the same or different, are selected, independently of oneanother, from hydrogen and straight-chain or branched alkyl having 1 to6 carbon atoms inclusive, its optical isomers, its possible N-oxides,and pharmaceutically-acceptable addition salts thereof with an acid orbase.
 2. A compound according to claim 1 which is selected from1-(N,N-dimethylaminoethylaminocarbonyl)-5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole, its N-oxides, and its pharmaceutically-acceptableaddition salts with an acid.
 3. A compound according to claim 1 which isselected from1-(N,N-dimethylaminopropylaminocarbonyl)5,6-dimethyl-9-hydroxy-6H-pyrido[4,3-b]carbazole, its N-oxides, and also its pharmaceutically-acceptableaddition salts with an acid.
 4. A compound according to claim 1 which isselected from1-(N,N-dimethylaminoethylaminocarbonyl)-5-methyl-9-hydroxy-6H-pyrido[4,3-b]carbazole, its N-oxides and also its pharmaceutically-acceptableaddition salts with an acid.
 5. A method for treating an animal or humanliving body afflicted with a disease requiring anti-tumor agentsinhibiting topoisomerases, resulting from cancer, comprising the step ofadministering to said living body an amount of a compound of claim 1which is suitable for alleviation of said disease.
 6. A pharmaceuticalcomposition useful in inhibiting topoisomerases which contains as activeingredient an effective amount of a compound according to claim 1, incombination with one or more pharmaceutically-acceptable excipients orvehicles.